A Comprehensive Data Processing Pipeline for Phosphoproteomics in Viral Infection Studies.

This protocol outlines a comprehensive methodology for investigating cellular responses to viral infections through quantitative global and phosphoproteome analysis. Phosphorylation, a crucial posttranslational modification, regulates various cellular processes and plays a key role in virus-host interactions. To study the phosphorylation events, we have developed an analysis pipeline, and the subsequent steps detail sample preparation techniques for obtaining high-quality protein samples suitable for phosphoproteomic analysis. These include lysing virus-infected cells, processing protein samples through reduction, alkylation, digestion, and desalting, and enriching phosphopeptides using CAE-Ti-IMAC microspheres. High-throughput DIA-PASEF MS-analysis using TimsTOF Pro 2 facilitates sensitive detection of phosphorylation events, enabling a comprehensive understanding of viral infection, replication, and cytotoxicity in the host cell. The protocol also integrates the DIA-NN program for processing and analyzing MS data. Overall, this protocol provides a robust framework for identifying potential therapeutic targets within these altered pathways for elucidating the molecular mechanisms underlying viral pathogenesis and host immune evasion.